Biochemistry tests

A
ALT - Alanine Aminotransferase

•  Liver disease
• Reference range: 20 - 60 IU/L

AST - Aspartate Aminotransferase (or SGOT)

• Cardiac or skeletal muscle disease

Albumin (Alb) - protein made by the liver.

• Liver disorder, Kidney disease, Nutritional status

ALP - Alkaline phosphatase test

• Liver disease and bone disorder, detect block bile duct.
• (GGT test is used to differentiate between liver disease and bone disorder)

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B
BUN - Blood Urea Nitrogen

• evaluate the kidney function

Bilirubin 

• Jaundice, liver disease, hemolytic anemia, blocked bile ducts
• 2 Forms : 
• Unconjugated bilirubin - converted from released heme of hemoglobin and carried to liver by protein.
• Total bilirubin level - Direct bilirubin level = Indirect level of unconjugated bilirubin.
• Conjugated bilirubin - sugar attached bilirubin which formed in liver.
• Direct bilirubin test: detect water-soluble form of conjugated bilirubin.

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C
Creatinine - waste product resulted from breakdown of creatine by the muscles.

• evaluate kidney function

CRP - (C-reactive protein)

• "Marker" of inflammation in body
• Monitor inflammatory disease activity, detect postoperative and neonatal infections and assess transplant rejection

CK - (Creatine Kinase)

• Skeletal and heart muscles disease
• Reference range: 30 - 225 IU/mL
• Isoenzyme:
• CK-BB  (CK-1)
• CK-MB (CK-2)
• More sensitive marker for heart injury because has lower basal level and a narrower normal range
• Reference range: 0 - 5 ng/mL
• CK-MM (CK-3)

Cholesterol

• Heart disease
• Reference range: below 200 mg/dL for adult

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G
GGT - gamma-glutamyl transferase

• elevated only when there is bile duct disease or liver disease.

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L
LDH - ( Lactate dehydrogenase)

• Cellular or tissue injury
• Reference range: 300 - 600 IU/mL.

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P
Potassium serum - abundant cation in intracellular

• Renal failure ....
• ( Other factitious causes: In vitro haemolysis, Aged specimen,... )  
• Reference range: 3.6 to 5.0 mEq/L.

Phosphorus

• diagnose or monitor treatment which will affect the phosphorus level.

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S
 Sodium serum - abundant cation in extracellular

• Hyponatremia ( Electrolyte abnormality/ Unbalanced serum osmolality)
• Reference range: 134 - 144 mEq/L.

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(Specimen requirement : blood within SST tube.)

For more information, log on to   http://clinlabnavigator.com    or   https://labtestsonline.org  .

# Case study 1

There is a patient who was suspected to be infected by Influenza virus.
The test which conducted yesterday showed negative result but the same test which done today shows positive result.
There are few reasons for this situation. The following are just some of the examples.

1. Laboratory -- The operator who conduct the test might have miss out some procedure during the first test.
2. Nurse -- The sample obtained is too little.
3. Manufacturer -- (Rare) The provided product is not functioning well.  

Problem Troubleshooting.

During the training session in Biochemistry Department, there are few problems encountered.

1.   Wash solution has finished while running a test on the sample.

• EFFECT :  The Analyser will not proceed if the problem is not solved
• SOLUTION : Replacement with new bottle of solution

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2.    Routine serial number do not match with the sample's after [Play] is pressed.

• SOLUTION :  Edit to the correct serial number and press [OK]

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3.    One requested test has been left out after the other tests were completed.

• EFFECT :  Prolonged TAT ( Turn around time )
• SOLUTION : Redo

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4.    Serum over spilled due to cap loosen.

• SOLUTION :  Wipe the contaminated surface area and spray with Distel.

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5.   Tubes arrived before the requisition forms reached our department.

• SOLUTION :   Check at the Procare software system.

• CASE :
  
  Today, there were 2 tubes found residing at biochemistry department for some times, but the requisition form is not yet reach in our hand.
  We could not assume that the two plain tubes have no investigation to be carried out, therefore I was advised by my mentor to check at the Procare system.
  
  After checking, we found that the first tube only need to undergo Dengue test which will be conducted at the Serology Department.
  Meanwhile, the second plain tube is involving several biochemistry tests, thus I will need to search the form for the second tube.
  
  The form was initially received by our laboratory at 8.13am but in the end we managed to get the form at 9.14am which indicates that the TAT has prolonged.
  
  Hence, I run the test immediately without any delay.

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6.   Sample Missing!

• CASE:
  
  The clerk came and asked for the patient test result, and we were so surprised that we could not find the patient name in our record. How come it went missing? Does it happen in every of the medical centre? Who is the culprit?

This is the culprit :

The shooter has faced problem when the form was sent.

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Blood Grouping

Human bloods are different from each others  due to the presence or absence of certain antigen and antibodies. Different combinations of these genetically inherited protein molecules provide different types of blood in every individual.

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ABO & Rhesus Grouping

• Forward - Blood cell + anti-A, anti-B, anti-AB, anti-D
• To test the presence of antigen
  
  
• Reverse - Serum + ABO red blood reagent, (Blood cell + anti-D )
• To test the presence of antibodies
  
  
  

2 Methods:

Tile method (30% suspension)

1. Place 1 drop of EDTA blood + 1 drop of the reagent at the particular square on the tile.
2. Fully mix the solution using a toothpick and agitate for reaction.

O Blood sure no agglutination because there is no "Anti-O"

Add 1 drop of reagent and 1 drop of blood.

Result ( the photo shown is "AB" positive)

Tube method (5% suspension)

• Washing *3 times* : (Reverse ✗ need)

1. Fill up the test tube with 1 drop of blood and top up with normal saline
2. Centrifuge at 3000 rpm for 1 minute
3. Dispose the supernatant.

• Resuspend the pellet in 19 drops of normal saline (5%) (Reverse ✗ need)
• Prepare and label 4 clean tubes with Anti-A, Anti-B, Anti-AB and Anti-D. (Reverse use ABO reagents)
• Withdraw 50μl of the sample solution and release them in each of the 4 tubes.
• Centrifuge the 4 tubes in 3000 rpm for 15 seconds. 
• Gently swirl the 4 tubes under light for result observation.
   

Result Interpretation:

• Positive result: Agglutination of RBC
• Negative result: The button dissolved followed by no agglutination. *even under microscope.*

• The Forward and Reverse test should have inverted/opposite result.
  Except : Baby  (Baby might not yet produce antibodies therefore the reverse result will be negative) 

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Du test and anti-CDE are performed when agglutination is not shown in Anti-D test.


3%~5% of antigen is the maximum amount which will form reaction with the particular amount of antibodies. Thus, 5% of suspension is used in the test.

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Reference:
http://www.edu.pe.ca/threeoaks/teacherpages/higginbotham/Biology%20521%20Webpage/resources.htm

Precaution to reduce the risk of making mistakes

Name
It's common that patient have similar name , therefore it will be better if the full name of patient is scanned and written down.

MRN/Patient registration number
In order to avoid making mistake, the MRN of the patient can be also counter checked along with the name.

Gender
Both age and gender of the patient is relevant to the reference.

Age
Both age and gender of the patient is relevant to the reference.

Sample Checking

• Integrity --- Colour of the serum , Sample haemolysis
• Sufficiency --- If too little serum, the probe will imbibe the gel beneath it.
• Fibrin and gel --- The fibrin and gel that being drawn will cause contamination of the tubing.

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Match the FBC with respective request form sounds simple but not actually. Why?
 ∵ There are few tips to be follow in order to avoid mismatch.

• Match by name
• Counter check with MRN ( Patient registration number )

                    EXAMPLE:

• Tan Ah Ning & Tan Ah Ming
• Subramaniam A/L Brabu & Subramaniam A/L Ramasamy

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Biochemistry test with Analyzer AU480 & Fibrinogen

It's a wonderful day, finally I managed to get a chance to operate the analyser under supervision.
Here is a few simple steps to follow.

Homepage → Rack requisition → Start Entry → Key in Sample ID → Profile ( select accordingly) → Demography ( Fill up the rest of the patient details) → Entry → Play → Start

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Before I load the sample to the rack, I had undergo the sample checking. And I found out that the serum is too little which might cause the probe of the analyser come into contact with the gel. Therefore I used an alternative method which shown by the photo below.

Low volume of serum is poured into a sample cup.

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Besides, today was so lucky that we managed to find some fibrin exist in the sample. The fibrin was so troublesome and had prolonged the Turn around time. We tried to rerun the test for three times every after the noticeable fibrin was removed.

Detected Fibrin

Method to remove : use a toothpick to swirl it out.

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Once the fibrin is not totally removed, the result will not be accurate or will not be completed.

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Factors that affect the accuracy of analysis result :

1. Sample integrity ( Haemolyse, Jaundice, Lipidemic ......)
2. Separating Gel (Insufficient serum) / Sample clot (Fibrin)
3. Reagent stability

Outsources the sample.

What is Outsource?
Outsourcing is one of the practice in the medical laboratory. It is like sending a test which is not doing in-house to the third party medical laboratory.


Where we outsource the sample?

• Gribbles
• Pantai Medical Centre
• Sime Darby
• DNA Lab
• ......

Reason for not running the in-house test.

• Cost ineffective
• Lack of Expertise
• Lack of facilities
• Second Opinion
• Not a common test

Du test.

D^u test 

• Confirmation for Rhesus-negative.

Procedure:

When the blood sample did not form agglutination with the Anti-D reagent, indicates that the sample is Rhesus-negative. 

1. Incubate the sample solution in water bath for 15 minutes.
2. Spin the test tube at 3000 rpm for 15 seconds.
3. If the result remain negative, wash three times at 3000 rpm for 1 minute each.
4. Dispose the supernatant and add 2 drops of AHG.
5. Spin the test tube at 3000 rpm for 15 seconds.
6. Look for any agglutination under microscope.

Why Sample Haemolyze?

What is Haemolysis?

When the serum shows reddish in colour and fibrin is produced, indicating the sample has haemolysed. This is due to the destruction of RBC membrane and release of the components within the cell.

(Left) Normal serum , (Middle) Haemolysed sample , (Right) Jaundice sample

Hemolyzed sample.

(Left)  4plus [++++] or more 

(Right) 3plus [+++]

Few causes of haemolyse:

1. In laboratory -- The tube is spun before the blood has clotted
2. Transportation -- Temperature, Duration, Storage
3. Blood drawing technique -- High force applied when the blood is drawn or placed into the tube.
4. Patient -- Fragile  RBC due to diseases or having treatment
5. Serum and RBC is not separated for too long.

How if the haemolysed sample is used?
The results of Potassium level, CKBM and few enzymes tests such as ALT, AST, ALKP and so on will not be accurate.


Example:
A patient sample which is haemolysed was tested and resulted in 5.8 for potassium value.
After the new sample from the same patient is retested, it shows 4.8 for the potassium value.

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More information:
http://www.bd.com/vacutainer/pdfs/techtalk/TechTalk_Jan2004_VS7167.pdf

Preparation of Blood Specimen

• Collection of specimen

Blood should be collected from patient slowly and smoothly without applying high force to avoid haemolysis. Blood which added into tubes containing additive should be inverted carefully to fully homogenize the solution.

• Spinning of Specimen

The blood in the serum separator tubes should be allowed to clot for at least 30 minutes in a vertical position prior to centrifugation. Short clotting times will result in fibrin formation, which may affect the complete gel barrier formation.

Preparation of W1 and Control

Preparation of W1

• 98ml of distilled water + 2ml of wash solution = refill the Wash 1 bottle
• Usage up to 2 weeks.

Equipment for preparation: Wash solution, Measuring Cylinder, Pipette, Distilled water.

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Preparation of Control

1. Fill the reagent bottle with the required amount of solution written on the external surface.
2. Fully mix the distilled water and reagent powder by inverting the bottle.
3. Fill each test tube with certain amount of reagent and cover with the parafilm.
4. Keep the completed test tubes in the freezer.

Equipment for preparation ( pointed by arrow ) : Parafilms, Scissor, Test tube, Beaker with Distilled Water, Pipette, and Reagent powder.

The Parafilms are cut incompletely for convenient purpose. 

Reagent Shortage.

Why it is important to check the reagent inventory?
Reason:  Because it involve the Turn around time.

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Not every analyser designed equally. What I want to share here is, some analyser is able to reload the reagent during half way of the process while some analyser will need to wait it completely stop functioning before we can reload the reagent.

For the analyser we use in our laboratory, we have to wait for it to stop completely before reload the reagent. Therefore it might prolong the TAT if the reagent inventory is not checked before running the test.

Example:

The on board urea reagent has only 20 tests remaining, while there are 30 samples to be investigated.
Hence, we still need another 10 or more units of the reagent volume.

If we have a reagent checking earlier, we can reload the reagent on the spot and will not be facing the reagent shortage during the process.

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How the reagent shortage affect the TAT?

Here is an example,
There are 30 samples in the first batch and still, there are new samples coming in.
We have no choice but to left the new samples aside to reload the new reagent.
The analyser will take around 20 minutes to stop completely before we are allowed to reload the empty reagent.


However when there is an emergency, you have to stop the analyser  by force in order to reload the new reagent. Even by doing so is able to shorten the TAT, it will still take some time to process. After we force the analyser to stop and reloaded the new reagent, we will need to update the inventory and run the calibration as well as the control based on the SOP ( Standard Operating Procedure ).


Suggestion/Solution

• Strongly recommended to check the reagent inventory before start the work.

TAT ( Turn Around Time )

What is TAT?
Turn around time is the amount of time that used to complete or fulfil a request. It can be taken as the indicator of the laboratory effectiveness.

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In the medical centre I am trained, they have a systematic way to track the TAT as shown in the photo below. We are able to know when the requisition form and sample have sent in and when the overall test has completed. Besides, the type and number of samples will also written on the form. Therefore, there will be a place to refer when the samples are suspected to be missing.

Cross Matching

Cross matching is conducted when there is a case for blood transfusion. The correct blood should be provided to the patient in order to avoid any transfuse reaction which may worsen the patient's health condition.

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Procedure:

• Blood Grouping (Both Forward and Reverse)
  *Tile method is used for rapid test during emergency*
  
• Centrifuge the plain tube.
  
• Take the requested unit of the relevant blood bag ( packed cell / whole blood )
  And cut 1 segment from each blood bag to release small amount of the blood to different labelled test tubes.
  Wash 3 times and make 5% suspension.
  
• Fill up the recipient card, place the card together with the blood bag and put them back to the fridge if the blood is not going to be collected immediately.

1. Add 100μl of patient's serum (from red-capped plain tube)  and 50μl of the Donor's red blood cells into a clean test tube.
2. Centrifuge for 15 seconds.
3. If no agglutination occurred, add 2 drops of LISS reagent and incubate at 37°C for 15 minutes.
4. Centrifuge for 15 seconds.
5. If no agglutination, wash 3 times  and add 2 drops of AHG reagent.
6. Centrifuge for 15 seconds.
7. View under microscope if agglutination is not observed visually.

Result interpretation: 

• No agglutination, add ccc ( Coomb's control cell/ checkcells ) to test the reliability of AHG
  *Should be have agglutination after centrifuge if the AHG reagent is valid *
• Agglutination indicating the donor's blood is not compatible with the patient's body. thus the blood must not be transfused.

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3 Phases

• RT (Room temperature) phase   -- to test when cold antibodies can function.  Eg. IgM ( Lewis....)
• LISS phase -- warm antibodies can function.  Eg:  1gG ( ABO )
• AHG phase

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Important Notes:

For newborn baby (less than 4 months), mother's blood/serum should be used for cross matching. Because, babies don't have antibodies yet.

Checklist

A checklist is created to avoid any important daily routines missed out.

These are the routines have to be done before proceed to the first batch of samples as the analyzer will not be able to stopped once the sample is run. 

AU480

• Quality Check
  

• Reagent Check
  
• ISE calibration check
  

• Check the sample probe if have any gel attached
• ISE reagent  
1. (REF) ISE reference
2. (M-STD) ISE MID Standard
3. (Buffer) ISE Buffer
• W1 & Clean is filled

Anti-CDE

Anti-CDE

• This test is performed due to the reason there are antigen and antibodies other than A,B,AB and D such as C, E, c, e.

Procedure:

1. Place 1 drop (50μl) of reagent and 1 drop of whole blood on a slide.
2. Mix well with the applicator stick.
3. Observe under microscope to see any agglutination occurred.

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* However, if Anti-D shows negative result while anti-CDE shows positive result, the patient will still be considered as Rhesus negative for safety purpose. *

Biochemistry Department.

Biochemistry
This department is mostly deal with those tests shown in the photo.

Different testing require different types of blood components. Therefore it is common when the nurse draw our blood for few times and insert into different tubes.

Most tests are performed using the serum. ( except those highlighted in the photo )

 So, what of the following component from the human body is used to perform the tests which are highlighted?

• A. Plasma
• B. Body Fluid.
• C. Urine
• D. Saliva

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The Answer is C. Urine

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More details about the functions of the tests can refer to https://labtestsonline.org/

Multiple roles in Biochemistry department

In these two weeks, I am assigned in biochemistry department. However, I have to take care other department as well. The workload of this department is more heavy compare to the Blood Bank.

In Blood bank, once a cross matching is done, means it is done. But in biochemistry department, when a test is completed, I'll still have to send the requisition form to other department which the test is still processing. For example, ESR, Urine department and so on...... 

Since I am holding the plain tube, I have the responsibility to run the test in Immunology and Serology if the test is not yet executed.

I have to make sure all the in-house test are done before I send out. Normally we out source those tests that are not running in-house because of few reasons.

• The test is not common
• Not cost effective
• Lack of facilities
• Lack of expertise
• Second opinion

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Once I finished all the biochemistry tests, I have to ensure all the tests requested are completed before sending to typing.   Besides, I must also counter check if all the requested tests are correctly completed.

• HbA1C
• Urine
• INR
• Dengue
• Influenza
• Mycoplasma
• ESR and so on......
• Sending out test

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Examples:

Case 1

All the biochemistry tests that requested had been completed which are the LA009 & LM001.

However, I must also send the plain tube/SST to Serology division to perform another 3 tests which are VDRL, ASOT and ANF/ANA.

Case 2

There is a 35 years old diabetes male patient whose urine glucose is 3plus [+++] and we correlate this result with his serum glucose. 

Urine test result

Serum glucose result

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Blood Bank

Not to lose out any knowledge I gained from the industrial training, I would like to share my experience during the training at the Blood Bank for the past 2 weeks before I went to biochemistry department.

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Blood Bank is the most important among all the departments as it involve blood collecting, processing, storing and providing.

• Blood Donation
• Blood Grouping
• Condition for Blood Storage
• Cross matching for Blood Transfusion

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My First Training Tray

Blood Bag.

Total, there are three types of blood bag, namely single blood bag, double blood bag and triple blood bag.

Needle on the blood bag that used to puncture into blood donor's vein.

• Single Blood Bag"
• It is used to collect whole blood only.

  

  
  

• Double Blood Bag"
• Can be used to collect whole blood and further separated into packed cells and plasma.

  

  
  

  
  
  
  
  
• Triple Blood Bag"
• The collected whole blood can be further divided into packed cells, platelets and plasma.

  

  
  





  








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The Potential of  Human Blood


• Whole Blood
  - From donation

  

  *Use plasma separator to divide the whole blood into packed cells,platelets and plasma.*

  
  
  
• Packed cell/ Red Blood cells
• Function: treat severe anemia by increase the amount of RBC in the patient.
• Storage: can be refrigerated for 42 days ( freeze for 10 years ) 

• Platelet concentrate. 
• Function: treat bleeding by increasing the platelet levels.
• Storage: 6 days in room temperature.

Placed on rotater to avoid agglutination takes place.

• Cryoprecipitate
• Function: treat fibrinogen deficiency
• Storage: 3 years in deep freezer.
  
  

• FFP ( Fresh Frozen Plasma )
• consists of all coagulation factors.
• Function: treat massive bleeding by correct the deficiency in coagulation factor.
• Storage: 3 years in deep freezer.

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Platelet Apheresis: Consists of 6 units of platelet concentrates from single donor.

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References:

1. http://ijtm.in/view_article.php?id=16
2. https://sites.google.com/site/vaaccs/home/vaaccs-blood-bank/blood-bags---what-do-i-need

My First Experience in Medical Laboratory.

Medical Laboratory/Pathological department

The place that I undergo my internship consists of few divisions :

1. Blood Bank
2. Biochemistry
3. Haematology
4. Serology
5. Microbiology
6. Immunology